Methods for culturing minimally-passaged fibroblasts and uses thereof

ABSTRACT

The present invention relates to novel methods of growing at least 100 million minimally-passaged fibroblasts from a small biopsy specimen. The invention includes methods wherein a small biopsy specimen is seeded directly into a large tissue culture flask, and passaged no more than three times.

FIELD OF THE INVENTION

The present invention relates to novel methods of culturing and growingautologous fibroblasts, and methods of use thereof to promote healingand regeneration of tissue in an animal.

BACKGROUND OF THE INVENTION

Fibroblasts are connective-tissue cells involved in tissue repair.Fibroblasts synthesize a variety of compounds, including collagens,glycosaminoglycans, reticular and elastic fibers, and glycoproteinsfound in the extracellular matrix. When a tissue is injured, nearbyfibroblasts migrate into the wound, proliferate, and produce largeamounts of collagenous matrix, which helps to isolate and repair thedamaged tissue. See, e.g., Alberts et al., Molecular Biology of the Cellp. 987, 2nd ed., (1992).

Co-owned patents U.S. Pat. Nos. 5,591,444; 5,858,390; 5,660,850;5,665,372; 6,432,710; and 6,878,383 (incorporated herein by reference intheir entirety), broadly describe the repair of skin, bone, and othertissues prior to the advent of the growing of fibroblast cultures drawnfrom biopsies of patients in need of tissue repair to effect suchrepair. In general, these patents also disclose compositions and methodsof growing and culturing passaged autologous fibroblasts and using suchfibroblasts to repair skin, bone, and other tissues.

Fibroblasts take significant time to grow to sufficient numbers, and thepassaging of cell cultures required to generate such numbers yieldsfibroblasts which may suffer from decreased viability and effectiveness.Multiply passaging fibroblasts also suffers drawbacks from increased useof materials, increased cost, and increased opportunities forcontamination of the cultures. Yet the art has not been able to generatesuch fibroblasts in sufficient numbers and effectiveness without amultiplicity of passages. The art, therefore, is in need of simplifiedmethods to generate such fibroblasts without the shortcomings ofmultiple passages. The present invention provides such a simplifiedmethod of growing such fibroblasts.

SUMMARY OF THE INVENTION

In a surprising discovery, the inventors herein have found that a small1-5 mm full thickness biopsy (on the order of a 3×3 mm circular shape)from a subject may be digested with a disassociating or digestiveenzyme, such as a collagenous enzyme (e.g., liberase) or trypsin to freedermal fibroblasts, which may be directly seeded into larger cultureflasks (in comparison to the typical known explant method) with anappropriate growth medium. The fibroblasts grow to confluence in largemembers after a short time of between about one and three weeks. Apassage to a larger flask, or an even larger multilayer culture stack,such as a 5- or 10-layer stack, with growth medium and routine additionof supplementary medium, leads to a harvest of autologous fibroblasts onthe order of 3×10⁸ or more cells. The cells may then be shipped directlyto the point of treatment location, either fresh or cryopreserved, ormay be cryopreserved, stored, and shipped at a later date.

Alternatively, the cells may be further cultured in additionalmultilayer culture stacks to generate larger quantities of cells, wheredesired. For example, the 3×10⁸ or more cells in a 10-layer stack may besplit into four additional 10-layer stacks, and cultured according tothe invention to generation more than 1×10⁹ cells.

The resulting fibroblasts grown by this method, which have beenminimally passaged, have improved viability and effectiveness comparedwith prior methods of preparing cultured autologous fibroblasts.

In general, the method of the invention comprises obtaining a sufficientnumber of autologous fibroblasts for treatment (at least 100 millioncells) by minimal passaging from a small biopsy specimen. The cellsobtained may then be used for repair of dermal defects by administeringthe autologous fibroblasts to a defect in skin, bone, or other tissue.The fibroblasts are prepared as a pharmaceutical composition asdescribed below, for direct injection, or may be delivered by othermeans such as topically. Preferably, the autologous fibroblasts areobtained from a tissue which is the same type of tissue as the defect tobe repaired. Additionally, the autologous fibroblasts may be cultured inthe animal's own serum, or in fetal bovine serum. The fibroblasts arepreferably passaged in culture at most three times, more preferably onlyone or two times. Between about 10 and 20 million autologous fibroblastsare generally administered per treatment.

The invention further provides a method of rendering the minimallypassaged dermal fibroblasts substantially free of immunogenic proteinspresent in the culture medium where desirable. The method comprisesincubating the expanded fibroblasts for a period of time inreduced-serum medium or serum-free medium supplemented with othernutrients.

The present invention further provides methods of correcting defects inan animal, such as a mammal, particularly a human. Desirably, the defectto be corrected is susceptible to healing upon administration ofautologous fibroblasts, such as defects in skin, bone, or otherconnective tissue.

The present invention provides methods of generating such minimallypassaged fibroblasts, and formulating a pharmaceutical composition forthe repair, augmentation, or regeneration of tissue, without surgery,wherein the composition is histocompatible with a subject, therebyavoiding elicitation of an immune response and inflammation in thetissues of the subject near the site of degeneration of tissue.

In one aspect, the present invention uses gentamicin as an antibacterialagent, as well as amphotericin B as a fungicide, during the culturestage. In another aspect, the invention avoids the use of antibiotics inthe subject, and hence prevents the emergence of antibiotic resistantpathogens and deleterious side effects associated with antibiotics inthe subject. In another aspect, the invention includes passagedautologous fibroblasts that can withstand resorption so that subsequentinjections are not needed, and to prevent the elicitation of an immuneresponse in the subject.

In one aspect, the present invention provides a method of correctingcosmetic and aesthetic defects in the skin of a subject by the injectionof a suspension of autologous dermal fibroblasts into the dermis andsubcutaneous tissue subadjacent to the defect. Typical defects that canbe corrected by this method include rhytids, stretch marks, depressedscars, cutaneous depressions of non-traumatic origin, scaring from acnevulgaris, and hypoplasia of the lip. Other tissues may also be repaired,such as vocal cords, oral mucosa, the gingival mucosa, or the palatalmucosa or skin, which has degenerated as a result of a disease ordisorder such as periodontal disease, trauma, dermatoses, recurrentaphthous stomatitis, and infections. Additionally, other damaged tissuemay be repaired using the fibroblasts generated by the method of theinvention, such as burns and burn scars.

The cells that are injected are cells that are histocompatible with thesubject, that is, they are autologous cells, that have been expanded byminimal passage in a cell culture system initiated by a biopsy specimen.

Thus, in one aspect, the invention is a method of generating at least100 million minimally-passaged fibroblasts from a small biopsy specimen,comprising the steps of:

-   -   a) digesting the small biopsy specimen with a dissociative or        digestive enzyme to form a cell suspension;    -   b) seeding the cell suspension into a first large tissue culture        flask;    -   c) culturing a fibroblast cell population in the first large        tissue culture flask through no more than three passages into        sequentially larger tissue culture flasks; and    -   d) harvesting the minimally-passaged fibroblasts.

In one aspect, the enzyme is trypsin, while in another, the enzyme is acollagenase enzyme. The collagenase enzyme may be liberase. In anotheraspect, the minimally-passaged fibroblasts have been passaged no morethan two times, or in other aspects, no more than once. In anotheraspect, the method of the invention generates at least 200 millionminimally-passaged fibroblasts are generated. In yet another aspect, themethod of the invention generates at least 300 millionminimally-passaged fibroblasts are generated.

In another aspect, the first large tissue culture flask is at least thesize of a T-125 flask, and preferably is a T-175 or a T-225 flask. Thesequentially larger tissue culture flasks may be a T-500 flask or amultilayer cell culture stack, such as a 5- or 10-layer cell culturestack.

In other aspects, the invention provides a method of treating a defectin the skin, bone, or other connective tissue of an animal, comprisingthe step of administering to the animal the minimally-passagedfibroblasts generated as described above, wherein the biopsy specimen istaken from the animal, i.e., the fibroblasts are autologous fibroblasts.

The defect may be selected from the group consisting of rhytids, stretchmarks, depressed scars, cutaneous depressions of nontraumatic origin,scarring from acne vulgaris, hypoplasia of the lip, vocal cords defects,defects of the oral mucosa, the gingival mucosa, or the palatal mucosaor skin, burns, and burn scars.

Administrations may each comprise 10-20 million minimally-passagedautologous fibroblasts, and may be injected into the defect area or areasubadjacent to the defect. In another aspect, the invention provides fortopical application of the minimally-passaged fibroblasts.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising and unexpecteddiscovery that biopsy derived fibroblasts may be seeded and cultured tonumbers sufficient for exogenous administration to a patient to promotehealing of tissue without requiring multiple passaging of the culturedcells. In the past, such biopsy-derived fibroblasts were required to beseeded at higher density in smaller tissue culture flasks, then multiplypassaged into sequentially larger flasks, in order to generate thedesired number of cells. The present invention solves the problem ofmultiple passaging by providing a method of seeding the biopsy-derivedcells directly into larger flasks, followed by only a minimum ofpassages to achieve the large number of cells needed for treatment ofdefects in skin, bone, and other connective tissue. As used herein, a“small biopsy” is one whose size is 1-5 mm thick, and between 4 and 12cm².

Methods of Generating Large Numbers of Fibroblasts with MinimalPassaging

Initially, a dermal fibroblast culture is initiated from a small 1 to 5mm full thickness biopsy specimen, generally taken from the skin, gums,or palate of the subject. Because of the phenomenon of allograftrejection, which is well known to transplantation surgeons andimmunologists, it is preferable that the cultured fibroblasts behistocompatible with the host. Histocompatibility can be ensured byobtaining a biopsy of the subject to be treated and culturing thefibroblasts from this specimen.

In brief, the method of the invention may be performed as follows:Before the initiation of the culture, the biopsy is washed several timesin a wash media comprising IMDM medium with antibiotic agents, such asgentamicin (antibacterial) at a concentration of between 20-40 mg/mL,preferably about 30 mg/mL, and amphotericin B (antifungal) at aconcentration of between 10-20 μg/mL, preferably about 15 μg/mL. Thebiopsy specimen is then digested using a solution of a dissociative ordigestive enzyme, and vortexed in an orbital shaker. In one embodiment,the enzyme is trypsin. In another embodiment, the enzyme is acollagenase enzyme, preferably liberase. Growth medium is than added toneutralize the enzyme (when such neutralization is necessary), and thecells are pelleted in a centrifuge. Preferably, growth medium comprisesIMDM (containing HEPES and L-glutamine and the aforementionedantibiotics) and 10% fetal bovine serum (PBS), although variations ingrowth medium will be appreciated by those of skill in the art.

The cells are resuspended in growth medium, and pipetted into a largetissue culture flask along with sufficient growth medium to keep allcells submerged. A “large” tissue culture flask is one which is at leastthe size of a T-125 flask, including T-125, T-150, T-175, T-225, T-500,and multilayer culture stacks. The flask is then incubated between35-39° C. with about 4-6% CO₂. Supplementation of the flask withadditional pre-warmed growth medium may be performed at intervals asneeded, usually about every 3-5 days. “Pre-warmed” medium is mediumwhich has been warmed after removal from refrigeration, though suchmedium need not be warmed to physiological temperatures. Whensupplementing with fresh media, it may be desirable to remove about halfof the existing media before adding in the fresh media, which may thenbe stored as conditioned medium for use elsewhere.

When the cells have reached about 40-100% confluence in the flask, theyare passaged into a larger tissue culture flask, such as a T-500 flaskor multilayer culture stack. This is accomplished by first removing thegrowth medium, which now comprises a variety of factors secreted fromthe growing culture (i.e., it is conditioned medium), which may bestored for use later. The flask is washed with phosphate buffered saline(PBS), then a solution of trypsin-EDTA is used to detach the fibroblastsfrom the wall of the tissue culture flask, according to procedures knownin the art. The detached fibroblasts are suspended in fresh growth mediato inactive the trypsin and transferred to a larger flask, such as aT-500 flask or multilayer stack. Again, fresh growth medium may be addedto the flask as needed.

If the first passage of cells was into a multilayer stack, or hasotherwise produced sufficient cells for treatment the cells areharvested as described below. Otherwise the same passaging procedure isused when the larger flask reaches about 95-100% confluence, to transferthe cells to a yet larger flask, such as a multilayer culture stack.Once again, additional growth medium may be added as needed andconditioned medium may be stored for later use.

When the cells in the multilayer culture stack have reached about95-100% confluence, they are harvested, generally yielding at least1.0×10⁸ cells, preferably at least 2.0×10⁸ cells, more preferably atleast 3.0×10⁸ cells. The cells may be cryopreserved as detailed below,or may be further cultured in additional multilayer culture stacks togenerate larger quantities of cells, where desired. For example, the3×10⁸ or more cells in a 10-layer stack may be split into fouradditional 10-layer stacks, and cultured according to the invention togeneration more than 1×10⁹ cells.

The cells may then be shipped directly to the point of treatmentlocation, either fresh or cryopreserved, or may be cryopreserved,stored, and shipped at a later date. Preferably, the cells will besuspended in 10-20 mL (this value is also variable depending on the cellpopulation at harvest, typically can be 10-20 mL) of freezing medium (asdescribed below), and transferred to freezing vials, 1.2 mL ofsuspension per vial. Each vial will thus contain about 2.2×10⁷ cells,sufficient for injection or other administration into a patient.Cryopreserved cells may be shipped frozen, or may be thawed, washed, andresuspended in appropriate media prior to shipping.

Numerous methods for successfully freezing cells for later use are knownin the art and are included in the present invention. The frozen storageof early rather than late passage fibroblasts is preferred because thenumber of passages in cell culture of normal human fibroblasts islimited.

The method of the invention as described in the above embodiment resultsin cells which have only been passaged only once or twice.

1.2 mL of fresh (or thawed previously frozen) suspension may be reseededinto a new large flask, or a 5- or 10-layer culture stack if more cellsare needed, or a stock of cells for the intended patient are to bemaintained, and the above procedure is followed until harvest of thestack. In this embodiment, the resulting stock of cells have beenpassaged three times.

As used herein, “minimally-passaged” fibroblasts refers to fibroblaststhat have been passaged a smaller number of passages in comparison toprior methods. For example, in generating 3×18⁸ or more cells in asingle 10-layer stack, the cells have been passaged no more than threetimes. In embodiments in which the cells have been further cultured intoadditional 10-layer stacks, the cells may have undergone additionalpassages, such as up to 4, 5, or 6 passages in generating 1×10⁹ or morecells.

The harvested minimally-passaged fibroblasts may be frozen in anyfreezing medium suitable for preserving fibroblasts. In one embodiment,freezing medium comprises by volume about 70% growth medium, about 20%FBS and about 10% dimethylsulfoxide (DMSO); however, variations in thecomposition and proportions in the freezing media will be appreciated bythose of skill in the art. However, in a preferred embodiment, no FBS isused in the freezing medium. Preferably the freezing medium comprises,by volume, 50% IMDM, 42.5% cryopreservation solution such as ProFreeze™,and 7.5% DMSO. DMSO may also be substituted with, for example, glycerol.Thawed cells can also be used to initiate new cultures by following themethods of the invention as described above, directly seeding in a10-layer culture stack, without the inconvenience of obtaining a secondspecimen.

Any tissue culture technique that is suitable for the propagation ofdermal fibroblasts from biopsy specimens may be used to expand the cellsto practice the invention as described above, maintaining a low numberof passages. Techniques well known to those skilled in the art can befound in R. I. Freshney, Ed., Animal Cell Culture: A Practical Approach(IRL Press, Oxford, England, 1986) and R. I. Freshney, Ed., Culture OfAnimal Cells: A Manual Of Basic Techniques, Alan R. Liss & Co., NowYork, 1987), which are hereby incorporated by reference.

The medium can be any medium suited for the growth of primary fibroblastcultures. The medium can be supplemented with human or non-human serumin an amount of between about 0.0% and about 20% by volume to promotegrowth of the fibroblasts. Higher concentrations of serum promote fastergrowth of the fibroblasts. Preferably, growth medium comprises IMDM(containing HEPES and L-glutamine) and 10% fetal bovine serum (FBS). Inanother embodiment, growth medium comprises glucose DMEM supplementedwith about 2 mM glutamine, about 110 mg/L sodium pyruvate, about 10%(v/v) fetal bovine serum and antibiotics, wherein the concentration ofglucose ranges from approximately 1,000 mg/L of medium to 4,500 mg/L ofmedium, and preferably is 4,500 mg/L.

Freshly harvested or thawed cells may be transported at 2-8° C., so longas they are utilized within 72 hours, preferably within 48 hours, andmore preferably within 24 hours of their suspension. The cells may besuspended in an appropriate transport medium, a physiological solutionwith appropriate osmolality, and may be tested for pyrogens andendotoxin levels. In another embodiment, the cells can be suspended inKrebs-Ringer solution comprising 5% dextrose or any other physiologicalsolution. In a preferred embodiment, the transport medium is DMEM.Cryopreserved cells are preferably transported on dry ice.

The volume of saline or transport medium in which the cells aresuspended is related to such factors as the number of fibroblasts thepractitioner desires to inject, the extent of the defects to thesubject's skin that are to be corrected, the size or number of thedefects that are to be corrected, and the urgency of the subject'sdesire to obtain the results of the treatment. Moreover, thepractitioner can suspend the cells in a larger volume of medium andinject correspondingly fewer cells at each injection site.

Preferably, about 10-20 million autologous fibroblasts are administeredper administration. Only viable fibroblasts should be administered.Fibroblasts generally remain viable for only about 24 hours outside ofculture when stored on ice and thawed. The number of autologousfibroblasts administered in any given administration may need to beadjusted up or down depending upon the potency of the fibroblasts (e.g.,collagen production), which may differ with the patient and tissuesources of the fibroblasts and which can be determined in accordancewith the assays set forth in Examples 2 and 3 or other assays as areknown in the art.

Administrations are repeated as necessary until the desired result isachieved. The timing of a repeat administration, if necessary, isdetermined by periodic assessment by a physician.

The fibroblasts can be administered with other active agents as desired.For example, the fibroblasts can be administered in conjunction withbasic fibroblast growth factor, which stimulates angiogenesis and ismitogenic for growth of keratinocytes and fibroblasts in vivo.

If desired, fetal or juvenile sources of fibroblasts can be used in thecontext of the present invention. Since fetal cells lack immunogenicdeterminants, they do not elicit a rejection response to the graft.

The minimally-passaged fibroblasts generated by the method of theinvention may be used for the repair, augmentation, or regeneration of avariety of tissues. Many such uses are disclosed in co-owned U.S. Pat.Nos. 5,591,444; 5,858,390; 5,660,850; 5,665,372; 6,432,710; and6,878,383, including the repair, augmentation, or regeneration of avariety of tissues. Thus, a pharmaceutical composition of the presentinvention can be injected into tissues of the subject, and thereby beused to correct defects in the skin, such as scars, wrinkles, laughlines, rhytids, stretch marks, depressed scars, cutaneous depressions ofnon-traumatic origin, acne scarring, or subcutaneous atrophy from acne,trauma, congenital malformation, or aging. Moreover, the invention canbe used to treat defects such a hypoplasia of the lips, labial folds,vocal cords, or defects in oral mucosa or palate, bone defects, or otherconnective tissue defects.

In one embodiment, then, the present invention provides a method forregenerating a subject's tissue that (a) has degenerated as a result ofa disease or disorder or (b) has a defect, comprising the steps ofproviding a pharmaceutical composition comprising autologous,minimally-passaged fibroblasts, identifying a site of tissuedegeneration, and injecting an effective amount of the composition intotissue at the site of the tissue defect or degeneration so that thetissue is augmented and regeneration of tissue is promoted.

In another embodiment, the method of the invention comprises (a)obtaining a sufficient number of autologous fibroblasts by minimalpassaging, and (b) administering the autologous fibroblasts to a defectin skin, bone, or other tissue. The fibroblasts are prepared as apharmaceutical composition, for direct injection, or may be delivered byother means such as topically. Preferably, the autologous fibroblastsare obtained from a tissue which is the same type of tissue as thedefect to be repaired. The fibroblasts are preferably passaged inculture less than three times, more preferably only one or two times.About 10-20 million autologous fibroblasts are preferably administeredper treatment.

The present invention, provides methods of generating such fibroblasts,and formulating a pharmaceutical composition for the repair,augmentation, or regeneration of tissue, without surgery, wherein thecomposition is preferably histocompatible with a subject, therebyavoiding elicitation of an immune response and inflammation in thetissues of the subject near the site of degeneration of tissue. Theinvention also may provide passaged autologous fibroblasts that canwithstand resorption so that subsequent infections are not needed, andto prevent the elicitation of an immune response in the subject.

In another embodiment, the present invention provides a method ofcorrecting cosmetic and aesthetic defects in the skin of a subject bythe injection of a suspension of autologous dermal fibroblasts into thedermis and subcutaneous tissue subadjacent to the defect. Typicaldefects that can be corrected by this method include rhytids, stretchmarks, depressed scars, cutaneous depressions of non-traumatic origin,scaring from acne vulgaris, and hypoplasia of the lip. Other tissues mayalso be repaired, such as oral mucosa, the gingival mucosa, or thepalatal mucosa or skin, which has degenerated as a result of a diseaseor disorder such as periodontal disease, trauma, dermatoses, recurrentaphthous stomatitis, and infectious. Additionally, other damaged tissuemay be repaired using the fibroblasts generated by the method of theinvention, such as burns and burn scars.

The cells that are injected are cells that are preferablyhistocompatible with the subject, that is, they are autologous cells,that have been expanded by minimal passage in a cell culture systeminitiated by a biopsy specimen. In a preferred embodiment, the injectedcells are dermal fibroblasts drawn from the subject to be treated.

Additionally, in some embodiments, the minimally-passaged fibroblastsmay be combined with acellular matrices and/or filler materials,depending on the intended treatment area, as described in the aboveco-owned patents. Other uses of cultured autologous fibroblasts known inthe art are equally amenable to using the minimally-passaged fibroblastsof the invention, such that they reap the benefit of minimal passagingof the fibroblasts.

Conditioned medium stored during the practice of the method of theinvention has many uses. For example, it may be used as a topicaltreatment for a variety of dermal defects, in conjunction with theadministration of fibroblasts grown by the method of the invention.Alternatively, the conditioned medium may be formulated into acomposition suitable for topical administration without any cells.

EXAMPLES

The following Examples serve to illustrate the present invention and arenot intended to limit its scope in any way.

Example 1: Generation of Minimally-Passaged Fibroblasts from SmallBiopsy Specimen Initiation of Culture

All procedures in this example were performed under sterile conditions.Initially, a dermal fibroblast culture was initiated from a small 1 to 5mm full thickness biopsy specimen from the skin of a human subject. Thebiopsy specimen was placed in a 50 mL conical tube and washed threetimes in a wash medium pre-warmed by incubation at 37.0±2.0° C. for 15to 30 minutes. The wash media comprised IMDM medium with gentamicin(antibacterial) at a concentration of 30 mg/mL and amphotericin B(antifungal) at a concentration of 15 μg/mL. For each wash, 20 mL ofwash medium was added to the 50 mL conical tube, and the biopsy wasmaintained submerged for 4-6 minutes. The wash media was then removed bypipette.

The washed biopsy specimen was then digested by pipetting 10 mL of apre-warmed solution of liberase enzyme for about 60 minutes. The conicaltube was then placed in an orbital shaker at 37.0±2.0° C. at 100 rpm forabout 120 minutes. The conical tube was then vortexed for 10 seconds.

10 mL of growth medium was pipetted to the tube to neutralize theliberase enzyme and suspend the cells, and the cells were then pelletedin a centrifuge at 150×g for 10 minutes at 5.0±3.0° C. with medium brakeand acceleration. Growth medium comprised IMDM (containing HEPES,L-glutamine, antibiotics gentamicin at a concentration of 30 mg/mL andamphotericin B at a concentration of 15 μg/mL), and 10% fetal bovineserum (FBS).

The supernatant was aspirated, and the cells were resuspended in 5.0 mLof growth medium. 40 ml of growth medium was placed in T-225 tissueculture flask and the resuspended cells were then pipetted into theflask. The flask was rocked gently to distribute the cells evenly overthe surface. The flask was then incubated at 37.0±2.0° C. with about4-6% CO₂. Supplementation of the flask with 20-50 mL additionalpre-warmed growth medium was performed at intervals as needed, aboutevery 3-5 days, and the removed conditioned media was stored for lateruse.

First Passage to Larger Flask

When the cells reached about 40-100% confluence in the flask, from about2-3 weeks, they were passaged into a T-500 flask. Generally, reachingthis level of confluence requires from about 2 weeks to about 30 days.The passage into a T-500 flask was accomplished by first removing thegrowth medium, which now comprised a variety of factors secreted fromthe growing culture (i.e., it was conditioned medium), which was storedfor use later. The flask was washed with about 30 mL of phosphatebuffered saline (PBS) pipetted into the flask and allowed to rest for4-6 minutes at room temperature. Then about 5 mL of a solution oftrypsin-EDTA (0.05% Trypsin/0.53 mM EDTA) was pipetted to the flask,which was incubated at room temperature for 4-6 minutes to detach thefibroblasts from the wall of the tissue culture flask. When 80-100% ofthe cells “rounded up”, having a round appearance, the sides of theflask were tapped to release the cells into suspension. About 15 mL ofpre-warmed growth medium was pipetted to the flask to neutralize theenzyme, and the detached fibroblasts were collected in a 50 mL conicalcentrifuge tube. An additional 10 mL of growth medium was added to theflask to collect any remaining detached cells, then added to thecentrifuge tube. The cells were pelleted, resuspended in 10 mL freshgrowth medium, and transferred to an upright T-500 flask pre-filled with80 mL of pre-warmed growth medium. The resuspended cells were pipettedto the T-500 flask and 10 mL of growth medium was used to rinse thecentrifuge tube, which was added to the flask as well. The flask waslaid flat to distribute cells and media to all three media layers of theflask. The flask was then incubated at 37.0±2.0° C. with about 4-6% CO₂.The T-500 flask was supplemented with additional growth media (about 50mL) as necessary, about every 3-5 days.

Second Passage to 10-Layer Cell Culture Stack

When the cells reached about 95-100% confluence in the T-500 flask,generally in only 3-5 days, they were passaged into a 10-layer culturestack. This was accomplished by first removing the growth medium, (nowconditioned medium), which was stored for later use. The flask waswashed with about 100 mL of phosphate buffered saline (PBS) pipettedinto the flask and allowed to rest for 4-6 minutes at room temperature.Then between about 10-20 mL of a solution of trypsin-EDTA similar tothat used previously was pipetted to the flask in an uptight positionuntil the layers equilibrated, then the flask was laid flat and gentlyrocked to ensure the entire growth surface was converged. The flask wasthen incubated at room temperature for 4-6 minutes to detach thefibroblasts from the wall of the tissue culture flask. When 80-100% ofthe cells “rounded up”, having a round appearance, the sides of theflask were tapped to release the cells into suspension. About 40 mL ofpre-warmed growth medium was pipetted to the flask while in the uprightposition to neutralize the enzyme, and the detached fibroblasts werepoured and/or pipetted into a 2 L media bottle pre-filled with 1300 mLof pre-warmed growth media. An additional 50 mL of growth medium wasadded to the flask to collect any remaining detached cells, then addedto the media bottle.

A 10-layer cell culture stack was prepared by replacing one of thestandard caps with a universal cap. The contents of the media bottlewere then slowly added to the 10-layer culture stack through a sterilefunnel in the port, while the opposite standard cap was loosened to ventthe stack, swirling the bottle at intervals during the pouring processto ensure capture of as many cells as possible. 100 mL of growth mediumwas added to the bottle to rinse its surface, and added to the 10-layerculture stack. The universal cap was then replaced with a solid cap. Theculture stack was tipped onto the side with the solid cap to allow mediato level across all layers of the culture stack, and then tilted towardsthe end without caps and placed flat again, then gently rocked. Finally,the solid cap was replaced with the original standard cap, and the10-layer culture stack was incubated at 37.0±2.0° C. with about 4-6%CO₂.

Again, fresh growth medium was added to the flask as needed byaspirating about half the volume of media and adding about 750 mL offresh growth medium in the same manner as described above. The aspiratedmedium was saved as conditioned medium for later use.

Harvesting of Minimally-Passaged Fibroblasts

When the cells in the 10-layer culture stack reached about 95-100%confluence, they were harvested, yielding about 3.0×10⁸ cells. First, 15mL of spent growth media was aspirated, to which about 5×10⁶ harvestedcells were added, to check for mycoplasma contamination. The remainingspent growth media was aspirated and saved as conditioned media. 600 mLof PBS was pipetted into the culture stack (300-600 mL may be used forthis purpose), replacing its filter vent caps with solid caps, and theculture stack was tipped allowing the PBS to wash all layers of thestack, which was then incubated for 2-3 minutes per wash. The PBS waspipetted or aspirated off after each rinse. 300 mL of Trypsin-EDTA(200-500 mL may be used for this purpose) solution was pipetted into theculture stack and evenly distributed therein. The culture stack was thenincubated for 4-6 minutes at 37.0±2.0° C.

When 80-100% of the cells “rounded up”, having a round appearance, thesides of the flask were tapped to release the cells into suspension.About 400 mL of pre-warmed growth medium was added and evenlydistributed therein to neutralize the enzyme, and the cell suspensionwas then transferred to two 500 mL conical tubes, about 350 mL in each.300 mL growth media as used to rinse the culture stack, which was thenevenly divided between the two 500 mL conical tubes. The cells were thenpelleted in a centrifuge at about 130-170×g for about 10 minutes at 5±3°C. The supernatant was aspirated. 30 mL of growth medium was used toresuspend the cells in one conical tube, then transferred to the othertube. 20 mL of growth medium was then used to rinse the first tube andthen added to the other tube. Additional growth medium was then added tothe tube containing the cell suspension to bring the total volume to 200mL. Quality control samples were taken at this time, though they may betaken at any other time during the process.

The cells were then pelleted, the supernatant removed, and the cellsresuspended in cold IMDM medium (5±3° C.) to a target concentration of4.4×10⁷ cells/mL (generally between about 5-10 mL). The cell suspensionis then stored in a refrigerator at 5±3° C.

Cryopreservation

In order to cryopreserve the cells, the cell suspension was diluted 1:1with an equal volume of freeze media comprising 85% ProFreeze™ and 15%DMSO. The volume of freeze media was prepared in a 15 mL tube, and thenslowly pipetted dropwise into the cell suspension, allowing the freezemedia to run along the side of the tube into the suspension. The tubewas then pulse vortexed for 5 seconds, wiped with 70% isopropyl alcohol,then pipetted into cryovials, 1.2 mL or 0.6 mL per vial depending onvolume requirements for testing and injection preparation. Thesuspension was swirled between each vial fill to ensure homogeneous cellsuspension during the filling process. Each vial contained about 2.2×10⁷cells, sufficient for injection or other administration into a patient.

Thawing and Final Preparation

Cells may be shipped fresh, or cryopreserved, or thawed fromcryopreservation. To perform a thaw and preparation for two productioninjection vials, 3.5 vials were warmed to 37.0±2.0° C. until almostcompletely thawed. The thawed suspension was pipetted into a 50 mLconical tube pre-filled with 17 mL of PBS. Each vial was rinsed withanother 1 mL of PBS which was then added to the tube. The cells werethen pelleted (150×g for 10 minutes at 5±3° C.). The PBS was aspirated,the cells were resuspended in 17 mL of DMEM, then repelleted. The DMEMwash was aspirated, and then the cells were resuspended in 3.0 mL ofDMEM. 0.1 mL of the suspension was removed for quality control analysis.An additional 5×10⁶ cells were collected and added to the previouslycollected 15 mL of spent media to submit for mycoplasma analysis. Thecells were now ready for shipping in a 2-8° C. cold pack system to thepractitioner for use. Alternatively, the cells may be shippedcryopreserved, to be thawed at the site where they will be used. Aftercryofreezing, vials are submitted to quality control for sterility andcell-based testing.

Example 2

Minimally passaged fibroblasts were generated as described in Example 1above, however, rather than cryopreserving the cells, they weretransferred to an additional four 10-layer stacks, and cultured asdescribed above for culturing cells in such stacks. When the cells inthe 10-layer culture stacks reached about 95-100% confluence, they wereharvested, yielding about 3.0×10⁸ cells per 10-layer stack, therebyproducing about 1.2×10⁹ cells. These cells were harvested as describedabove, ready for cryopreservation or for shipping as fresh cells fortreatment of patients.

The present invention is not to be limited in scope by the specificembodiments described above, which are intended as illustrations ofaspects of the invention. Functionally equivalent methods and componentsare within the scope of the invention. Indeed, various modifications ofthe invention, in addition to those shown and described herein, willbecome apparent to those skilled in the art from the foregoingdescription. Such modifications are intended to fall within the scope ofthe appended claims. All cited references are, hereby, incorporated byreference.

We claim:
 1. A method of preparing a composition comprising a cell-freefibroblast conditioned medium comprising the steps of: obtaining a smalltissue biopsy specimen from a human; digesting the biopsy specimen witha dissociative or digestive enzyme to form a fibroblast cell suspension,wherein the biopsy specimen is no more than 5 mm in thickness and notmore than 12 cm² in area; seeding the fibroblast cell suspension into afirst tissue culture flask; culturing the fibroblast cell suspension inthe first tissue culture flask through at most three passages into asequentially larger tissue culture flask; harvesting the fibroblastconditioned medium from the cell suspension during the culturing step,wherein the fibroblast cell suspension comprises at least 3×10⁸ cells atharvest; and formulating the conditioned medium into the composition. 2.The method of claim 1, the method further comprising administering acomposition to the human.
 3. The method of claim 2, wherein thecomposition is administered topically.
 4. The method of claim 2, whereinthe composition is injected into a defect in skin, bone, or otherconnective tissue of the human or an area subadjacent to the defect. 5.The method of claim 4, wherein the defect is selected from the groupconsisting of rhytids, stretch marks, depressed scars, cutaneousdepressions of non-traumatic origin, scarring from acne vulgaris,hypoplasia of the lip, vocal cord defects, defects of the oral mucosa,the gingival mucosa, or the palatal mucosa or skin, burns, and burnscars.
 6. The method of claim 1, wherein the enzyme is a collagenase. 7.The method of claim 6, wherein the collagenase is liberase.
 8. Themethod of claim 1, wherein the enzyme is trypsin.
 9. The method of claim1, wherein the first tissue culture flask is a T-125 flask, T-175 flask,or T-225 flask.
 10. The method of claim 1, wherein the sequentiallylarger tissue culture flask is a T-500 flask or multi-layer culturestack.